Amino acid residues in both the protein splicing and endonuclease domains of the PI-SceI intein mediate DNA binding.
A structure-based model describing the interaction of the two-domain PI-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PI-SceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4- to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active. Alanine substitution at two positions in domain I reduces overall binding >60-fold by perturbing the interaction of PI-SceI with the minimal binding region. Conversely, mutations in domain II have little effect on binding, reduce binding to the cleavage site region only, or affect binding to both regions. Interestingly, substitutions at Lys301, which is part of the endonucleolytic active site, eliminate binding to the cleavage site region but permit contact with the minimal binding region. This experimental evidence demonstrates that the protein splicing domain as well as the endonuclease domain is involved in binding of a DNA substrate with the requisite length.
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Center for Macromolecular Design, Institute of Biosciences and Technology, Houston, Texas 77030, USA.
Crist
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Yen
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Duan
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Quiocho
F A
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Gimble
F S
FS
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D016296:Mutagenesis; D011485:Protein Binding; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
D000596:Amino Acids; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific